In the light of the COVID-19 pandemic, the development of sensitive, specific, and cost-effective diagnostic tools has been highlighted. Among various diagnostic methods, molecular diagnostics including real-time polymerase chain reaction (qPCR) is considered to be the golden standard in the current state of the art due to its high sensitivity, specificity, and quantitation. Despite the advantages of qPCR, it is vulnerable to PCR inhibitors and relies on relative quantitation based on a standard curve, leading to false-negative diagnostic results. As an alternative, droplet digital PCR (ddPCR) has been highlighted for its higher sensitivity compared to qPCR and absolute quantitation that does not require a standard curve for each diagnostic run. In spite of the strengths of ddPCR, the high cost of ddPCR equipment and the complex and cumbersome sample pretreatment procedures significantly hinder its direct application to diagnostics. To overcome such limitations, a number of microfluidic devices have been introduced to perform ddPCR in microfluidic chips. Although several microfluidic devices have demonstrated comparable performances to that of the conventional benchtop ddPCR, their complicated operation principles such as centrifuge and external energy requirement and long sample preparation process still need to be improved. In this study, we developed a push-button activated microfluidic device based on polydimethylsiloxane using indirect pressurization for sample preparation in ddPCR. We have adopted a previously developed pushbutton-activated microfluidic device for nucleic acid extraction and droplet generation into a single microfluidic device to efficiently perform integrated sample preparation steps. A conventional solid-phase extraction method was adopted for nucleic acid extraction to capture and release DNA from silica microbeads. A reciprocating flow was generated inside a microfluidic device to increase the chance of DNA binding to silica beads. The generated reciprocating flow was further characterized to confirm that no fluidic loss occurred during the oscillation of the fluid. Subsequently, the extracted DNA was compartmentalized into droplets based on a conventional T-junction channel. The size of droplets was controllable by adjusting the dimension of the droplet generation channels. To examine the validity of monodisperse droplet generation, droplets from various geometries of the droplet generation channel were generated and the size distribution of droplets from each geometry was calculated. The proposed device significantly reduced the time for sample preparation by 75% compared with the time for sample preparation in the conventional benchtop method. To check the validity of the integrated device, hepatitis B virus standard DNA was first extracted, mixed with digitized PCR mixtures, and compartmentalized into droplets in a single device. The generated droplets underwent a thermal cycle for their fluorescence reading by the conventional fluorescence signal analyzer. We believe that the proposed device has the potential in simplifying and reducing the cost of sample preparation steps in ddPCR.
코로나19 사태로 인해 전세계적으로 민감하고 특이적이며 비용 효율적인 진단 방법이 주목받고 있으며, 다양한 진단 방법 중에서도 분자 진단 중 real-time PCR (qPCR)은 고민감도와 높은 특이도 그리고 정량이 가능하다는 점에서 오늘날 분석 표준으로써 작용하고 있다. 그럼에도 불구하고 qPCR은 PCR 억제제에 취약하며 표준 곡선이 필요한 상대 정량에 의존한다는 한계점을 지니고 있어 위음성의 결과를 초래할 수 있다. 대안으로서 droplet digital PCR (ddPCR)은 qPCR 대비 더 높은 민감도를 보이며 절대 정량이 가능하다는 장점을 지니고 있지만 ddPCR 장비의 높은 가격과 복잡하고 번거로운 시료전처리 과정은 ddPCR을 진단 분야에 적용하는 데 있어 큰 한계점으로 작용하고 있다. 본 논문에서는 polydimethylsiloxande (PDMS)로 이루어진 버튼 기반 미세유체소자에서의 가압 방식을 통한 ddPCR 시료전처리 연구를 진행하였다. 기존에 개발되었던 핵산 추출과 액적 생성 미세유체소자 각각을 하나의 미세유체소자로 통합하여 더 효율적인 시료전처리 과정을 수행하였다. 핵산 추출 방법으로는 통상적인 고상 추출 방법을 채택하였고, 추출된 핵산은 액적으로 구획화 하였으며 단분산 액적 생성을 검증하기 위해 액적의 크기를 측정하였다. 제안된 소자는 통상적인 방법인 시료전처리 과정에서 걸리는 시간보다 무려 75% 단축시킬 수 있었다. 개발된 소자의 타당성을 확인하고자 B형 간염 바이러스의 표준 핵산을 추출 후 PCR 혼합물과 혼합 후 액적으로 구획화 하였고, 구획화된 액적은 형광 신호를 위해 열주기를 거쳤으며 이후 통상적인 형광 신호 분석기로 형광 신호를 측정할 수 있었다. 본 논문에서 제시된 소자는 ddPCR의 시료전처리 과정을 좀 더 간편화하고 가격을 낮출 수 있을 것이라 기대 된다.
